The non-pathogenic yeast Candida (Starmerella) bombicola ATCC 22214 (CBS 6009) is commercially applied for the production of sophorolipids. These glycolipid biosurfactants are constituted of a sophorose head group (2-O-β-D-glucopyranosyl-D-glucopyranose) attached to a (sub)terminal hydroxylated C18 or C16 fatty acid by a glycosidic linkage between the anomeric C-atom of the sugar and the hydroxyl group of the fatty acid. Sophorolipids are typically produced by fermentation in the presence of a hydrophobic carbon source and are always constituted of a mixture of structurally related molecules with variation in 1) degree of fatty acid saturation (saturated, mono-unsaturated or di-unsaturated), 2) presence or absence of acetyl groups at C6′ and/or C6″ atoms, 3) lactonization between the carboxyl end of the fatty acid and either the C4″, C6′ or C6″ atom of the sophorose group resulting in a lactonic sophorolipid or absence of this lactonization, resulting in an open or acidic sophorolipid, 4) fatty acid chain length and 5) (ω) or (ω-1) hydroxylation of the fatty acid (Asmer et al., 1988).
Due to this structural variation, sophorolipids show many interesting applications in a wide range of industrial fields (Banat et al., 2010; Franzetti et al., 2010; Kralova and Sjoblom, 2009; Mulligan, 2009). Since structural composition is reflected in the physico-chemical properties, several industries are particularly interested in specific structural variants. Lactonized sophorolipids have different biological and physicochemical properties compared to acidic forms. In general, lactonic sophorolipids have better surface tension lowering and antimicrobial activity, whereas the acidic ones display a better foam production and solubility (Lang et al., 2000)
On the other hand, acidic sophorolipids have been used as starting molecules for the synthesis of, e.g., dispersible nanoparticles (Kasture et al., 2007) and glycolipid derivatives (Azim et al., 2006; Zerkowski et al., 2006) or have served as source molecules for the production of glucolipids and specialty fatty acids (Rau et al., 2001; Saerens et al., 2009), which are on their turn used for synthesis of polymers (Zerkowski et al., 2007) or precursors for plastics and flavors (Rau et al., 2001). Lactonization reduces the rotational freedom of the molecule; dominance of this type of sophorolipids very often results in the formation of crystals instead of the more common viscous oil, rendering the sophorolipids relatively easy to isolate.
To date, it is impossible to efficiently produce 100% acidic sophorolipids by fermentation with C. bombicola ATCC22214.
It is known that the lactonic/acidic balance is to a certain point influenced by fermentation conditions such as the level of yeast extract, oxygen supply and the provided lipidic substrate (Garcia-Ochoa and Casas, 1999). However, it is unclear what mechanism exactly determines the degree of lactonization.
In a typical C. bombicola fermentation on glucose and oleic acid, 62% of the sophorolipids are composed of diacetylated lactonic forms, 4% is composed of monoacetylated lactonic forms, and 4% is composed of unacetylated lactonic forms, while the other compounds are constituted of 1′,6′ lactones and 1′,6″ lactones (4%), acidic sophorolipids (8%), and other lipids at the end of the cultivation period (Asmer et al., 1988). Hu and Ju (2001b) observed a maximum relative percentage of lactonic forms of 50% using soybean oil and 80% using hexadecane.
In another experiment where palm esters were used, 79.1% occurred in the lactonic form, but when sunflower oil was applied, only 55.6% lactonic sophorolipids were retrieved (Davila et al., 1994). When in a mixed carbon source fed-batch fermentation only oil was added in stationary phase, 13% of the sophorolipids were in the lactonic form (87% acidic); but when a mixture of oil and glucose was fed in stationary phase, 65% were in the lactonic form and, moreover, a much higher production was achieved (Davila et al., 1997). Yeast extract concentration and presence of citric acid influence the ratio of lactonic to acidic sophorolipids too: when yeast extract concentration was 1 g/L, 65% of the sophorolipids occurred in the lactonic form; but when the concentration was increased to 20 g/L, only about 2% were in the acidic form. However, yeast extract concentration is negatively correlated to sophorolipid yield; indeed, with 1 g/L, 76 g/L sophorolipids were obtained at the end of the cultivation period, while this was only 13 g/L for the set-up with 20 g/L (Casas and Garcia-Ochoa, 1999). Addition of 5 g/L of citric acid to the medium increased the percentage of lactonic forms in the sophorolipid mixture of Candida apicola (Hommel et al., 1994).
The presence of citrate in the cultivation medium was described to be absolutely necessary to obtain lactonized sophorolipids. It was suggested by Stüwer et al., (1987) that the effects seen for citrate are probably just an effect of the buffering action of the conjugate base of a weak acid. Low pH values lead to the production of acidic sophorolipids (=SLs) and the buffering effect of citrate would thus favor the formation of lactonic SLs. Citrate, on the other hand, is a chelating agent, and metal ions like zinc or calcium could be necessary for the action of a hypothetical enzyme responsible for ring opening. The presence of citrate would in this hypothesis prevent ring opening and, as such, favor the predominance of lactonic SLs in the cultivation medium. Another possibility is that citric acid has some kind of regulatory effect as was described for the regulation of lipid accumulation in oleaginous yeasts (Evans and Ratledge, 1985).
The exact ratio of lactonic to acidic sophorolipids further changes during cultivation with typically more lactonic forms after prolonged incubation times (Casas and Garcia-Ochoa, 1999; Hu and Ju, 2001b).
In brief, one can state that dominance of the acidic forms in the sophorolipid mixture produced by wild-type C. bombicola is linked to cultivations under suboptimal conditions and consequently always results in lower yields as compared to the standard conditions described in literature.
Recently, other yeast species producing sophorolipids similar to those of C. bombicola were described. In some cases, the lactonic:acidic ratio is different when compared to the ratio for C. bombicola obtained under the same cultivation conditions.
In the sophorolipids produced by C. batistae, for instance, the acidic forms make up about 60% of mixture compared to 34% for C. bombicola (Konishi et al., 2008). The same trend is observed for C. riodocensis, C. stellata and Candida sp. NRRL Y-27208, which produced predominantly free acid sophorolipids when compared to C. bombicola and C. apicola. Furthermore, Imura et al., (2010 and JP2008247845) isolated the strain Candida floricola TM 1502, which preferentially gives diacetylated acid-form sophorolipids without including lactone-form sophorolipids. The latter strain is the only one described to produce 100% acidic sophorolipids. However, total sophorolipid production is significantly lower when compared to the amounts obtained with C. bombicola, hampering the industrial application of this strain.
The only way to obtain acidic sophorolipids in high purity and quantity is via the chemical conversion of natural C. bombicola sophorolipid mixtures to acidic products by alkaline hydrolysis. By this conversion, however, all other ester-bonds will be hydrolyzed as well, resulting in the removal of any present acetyl groups and the consequent production of only acidic unacetylated sophorolipids.
Until now, it remained unclear how and when lactonization occurred. On the one hand, spontaneous lactonization of aqueous solutions of acidic non-acetylated sophorolipids has been observed, suggesting a spontaneous process. Furthermore, the presence of citrate is claimed to influence the lactonic:acidic ratio (Hommel et al., 1994; Stüwer et al., 1987). On the other hand, Hommel et al. (1994) suggest the involvement of a cell wall-bound lipase in the lactone formation. In this regard, it was demonstrated that the commercial Novozyme 435 or lipase B from Pseudozyma antarctica (former C. antarctica) can, under rather non-physiological laboratory conditions, lactonize an acidic unacetylated sophorolipid ester at the 6″-position in anhydrous tetrahydrofuran (Bisht et al., 1999; Nunez et al., 2003).
Furthermore, Van Bogaert et al. (2011) recently reviewed the microbial synthesis of sophorolipids. In this review, the authors indicate that it is believed that a specific lactone esterase mediates lactonization of sophorolipids in C. bombicola, but that no such enzyme has been identified.
Taken together, it is clear that identification of an alternative enzyme responsible for an efficient lactonization would offer great potential in the control of structural variability in the sophorolipid production and production of a specific sophorolipid mixture.
However, there was until now not a single hint in the art indicating if and where such an enzyme could be found in the genome of C. bombicola, and/or what the role of citrate is, and/or if multiple enzymes or a single enzyme are/is fully responsible for the lactonization process.